Ash dieback is an emerging disease of Fraxinus excelsior in Germany. To date, economical damage is significant in nurseries, which also contribute towards spread of the disease, but damage to forests is increasing. The study presents the results of mycological and histological investigations on three hundred 3-year-old nursery ash saplings. The infection rate by the causative pathogen was determined for bark, outer and inner xylem, the pith and also separately for the above-ground portion and root system of the plants. The invasion and colonization strategy of the fungus in the woody stem was examined. In addition, the presence of soil-borne Oomycetes as possible primary or accompanying causal organisms was investigated. The results verify the dominant role of Chalara fraxinea as a causal agent of ash dieback and rule out the role of Oomycetes in the disease process. We conclude that C. fraxinea is not primarily endophytic in nature and spreads very effectively in the central stem tissues, which enables colonization of the woody stem in all three dimensions. Infections arising in the upper part of plants can thus spread extensively to lower parts.
The survival of two hygienically relevant bacteria, Escherichia coli pIE639 and Enterococcus faecium, was followed on wooden sawdust of seven different European woods (pine, spruce, larch, beech, maple, poplar, and oak) versus polyethylene chips by using cultivationdependent and molecular-based methods in parallel. The survival of the bacteria on wood was dependent on various factors such as the wood species, the type of the inoculated bacterium, the ambient temperature, and humidity. The bacterial titre decreased fastest on pine followed by oak compared to the other woods and plastic. Cultivation-independent analysis employing DNA extraction, Southern blot hybridisation, and PCR-based detection of marker genes of the test bacteria confirmed this result. The decline in bacterial numbers correlated with the decrease of bacterial DNA in the samples. Amounts of DNA of E. coli and E. faecium recovered from pine and oak-wood sawdust were generally lower compared to the other woods and plastic.The presented study shows that pine and oak exhibit substantially better hygienic performance than plastic and indicates an antibacterial effect caused by a combination of the hygroscopic properties of wood and the effect of wood extractives.
The survival of bacteria on and in different wooden and plastic boards was examined by microbiological methods. Wood of different tree species and polyethylene were inoculated with Escherichia coli pIE639 and Enterococcus faecium as hygienically relevant test bacteria. The development of the bacterial titer was evaluated by culturing on agar contact plates and investigating wood shavings. Survival of the test bacteria depended on different factors such as tree species, the initial inoculum size and the characteristics of the inoculated strain. The bacterial titer decreased fastest on pine compared to other woods (spruce, beech, poplar) and plastic. After bacterial infestation only pine wood was germ-free at the surface and in the inner structure after a few hours. The survival of the bacteria on poplar and beech was comparable to their survival on plastic. The study indicated that an antibacterial effect of wood, especially for pine, is caused by the hygroscopic properties of wood and the wood extractives. The antibacterial effect of pine wood was not influenced by the storage time of the wood following harvest or the functional condition of the wood up to a germ load of 10 8 CFU/cm 2 E. coli pIE639. Beinflussen Holzbretter das Überleben von Bakterien? Das Ü berleben von Bakterien auf und in verschiedenen Brettchen aus Holz und Kunststoff wurde mit mikrobiologischen Methoden untersucht. Verschiedene Holzarten und Polyethylen wurden mit Escherichia coli pIE639 und Enterococcus faecium als hygienisch relevante Testbakterien beimpft. Die Entwicklung des Bakterientiters ist durch Abklatschproben und Untersuchung von Hobelspänen verfolgt worden. Das Ü berleben der Testbakterien war dabei von verschiedenen Faktoren abhängig, wie z.B. von der Holzart, der Animpfdichte und der Art des inokulierten Bakterienstamms. Der Bakterientiter nahm im Vergleich zu anderen Holzarten (Fichte, Buche, Pappel) und Kunststoff am schnellsten auf Kiefernholz ab. Nur Kiefernholz war innerhalb weniger Stunden an der Oberfläche und im Innern des Holzes keimfrei. Das Ü berleben der Bakterien auf Pappel-und Buchenholz war mit dem Ü berleben der Bakterien auf Kunststoff vergleichbar. Die Studie deutet auf eine antibakterielle Wirkung von Holz hin, besonders von Kiefernholz, aufgrund der hygroskopen Eigenschaften von Holz und der Wirkung von Holzinhaltsstoffen. Die antibakterielle Wirkung von Kiefernholz wurde durch die Dauer der Lagerung nach der Holzernte und den Gebrauchszustand der Brettchen bis zu einer Keimbelastung von 10 8 CFU/cm 2 E. coli pIE639 nicht beeinflusst.
Ozone and light effects on endophytic colonization by Apiognomonia errabunda of adult beech trees (Fagus sylvatica) and their putative mediation by internal defence compounds were studied at the Kranzberg Forest free-air ozone fumigation site. A. errabunda colonization was quantified by "real-time PCR" (QPCR). A. errabunda-specific primers allowed detection without interference by DNA from European beech and several species of common genera of plant pathogenic fungi, such as Mycosphaerella, Alternaria, Botrytis, and Fusarium. Colonization levels of sun and shade leaves of European beech trees exposed either to ambient or twice ambient ozone regimes were determined. Colonization was significantly higher in shade compared to sun leaves. Ozone exhibited a marginally inhibitory effect on fungal colonization only in young leaves in 2002. The hot and dry summer of 2003 reduced fungal colonization dramatically, being more pronounced than ozone treatment or sun exposure. Levels of soluble and cell wall-bound phenolic compounds were approximately twice as high in sun than in shade leaves. Acylated flavonol 3- O-glycosides with putatively high UV-B shielding effect were very low in shade canopy leaves. Ozone had only a minor influence on secondary metabolites in sun leaves. It slightly increased kaempferol 3- O-glucoside levels exclusively in shade leaves. The frequently prominent hydroxycinnamic acid derivative, chlorogenic acid, was tested for its growth inhibiting activity against Apiognomonia and showed an IC50 of approximately 8 mM. Appearance of Apiognomonia-related necroses strongly correlated with the occurrence of the stress metabolite, 3,3',4,4'-tetramethoxybiphenyl. Infection success of Apiognomonia was highly dependent on light exposure, presumably affected by the endogenous levels of constitutive phenolic compounds. Ozone exerted only minor modulating effects, whereas climatic factors, such as pronounced heat periods and drought, were dramatically overriding.
Controlled inoculation of spruce seedling needle crowns and of shoots of 4-year-old spruce trees by Sirococcus conigenus led to disease symptoms (discoloration and necrosis) and to the induction of phenolic metabolites. Even upon complete infection, as proved by reisolation of the pathogen from inoculated seedlings, only 40% of the plants developed visible disease symptoms after 38 days. A Sirococcus-specific polymerase chain reaction (PCR) primer pair, SIRO1 and SIRO6, was designed based on sequences of a RAPD fragment. The primer pair permitted the detection of 1 pg fungal DNA (10-40 genomes) in 1 mg fresh weight spruce tissues (needles, bark, wood), regardless of visible disease symptoms. The amounts of the major phenolic compound of spruce needles, catechin, increased significantly in all of the five spruce provenances as a response to inoculation with Sirococcus. The second major phenolic compound, picein, increased in three of the provenances, whereas the remaining two had high concentrations to begin with and showed no reaction. Minor phenolic compounds increased in response to infection regardless of provenance. In a preliminary field study, Sirococcus infection of spruce was detectable by PCR even in the presence of massive infection by other fungi, such as Rhizospaera spp. and Lophodermium spp.
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